JPS61120058A - Method and instrument for analyzing intended component - Google Patents

Abstract

PURPOSE:To analyze a physiological material conjugated with protein, etc. in a short period with high accuracy by controlling the pH of the soln. of a material to be detected conjugated with the intended component and protein, electrifying said material and adsorbing such material to a macro-network ion exchange resin then eluting only the intended component. CONSTITUTION:A sample soln. of the material to be detected consisting of the protein conjugated with the component to be analyzed, for example, serum, etc. is adjusted in pH to decrease the pH by the protein. Such sample soln. is supplied from an injection part 2 into a column 3 packed with the cation exchange resin and in the case of increasing the pH, anion exchange resin having respectively the macro-network structure. The elutes of which the pH is changed according to the intended component are thus supplied from respective storage tanks 8A, 8B, 8C to the column 3 adsorbed with the protein in the above-mentioned manner. The eluted component is separated by a column 4 and the absorbancy of, for example, the separated component is measured by an analyzing instrument 6 the result thereof is displayed on a recorder 7. The protein removal is thus quickly and substantially executed and the exact analysis is executed.

Description

[Detailed Description of the Invention] [Field of Application of the Invention] The present invention provides an analysis method for separating and analyzing target components such as low molecular weight physiological substances, drugs, and their metabolites bound to proteins in solution from proteins. and its analysis device.

[Background of the invention]

Conventionally, to analyze target components bound to proteins, there are biological methods that do not remove the proteins contained in the test solution, and methods that are performed after removing the proteins by physical or chemical manipulation. There are physical and chemical methods to do this.

The above biological method analyzes the target component using an enzyme that specifically reacts with the target component, but there are problems with the stability of the enzyme), the relimitability of the analysis results, and the manipulation of the analysis. There are many disadvantages in terms of gender etc.

In addition, the above chemical analysis method attempts to separate the target component from the target component when hydrochloric acid is applied to the target component bound to the protein to denature and precipitate the protein, and then perform analysis on the target component. In this analysis method, when the protein is precipitated, there is a risk that the target component will also be precipitated together with the protein, which poses a problem in terms of the accuracy of the analysis data.

Therefore, as a physical method, proteins are charged with acids or bases, and the charged proteins are passed through an ion exchange resin and adsorbed. There is also a method that removes only the target component from proteins by changing the pH conditions after adsorption.

However, with this physical method, there is a possibility that the protein will be eluted together with the target component. Therefore, considering these problems, Japanese Patent Application Laid-Open No. 58-2230614+, Japanese Patent Laid-Open No. 58-22
As disclosed in No. 3062, a column filled with protein-coated separation material is used, and an analysis sample is passed through this column, and target components bound to proteins are adsorbed, while proteins are not adsorbed. There is also a method of removing protein.

However, although the conventional example described above is excellent in the recovery rate of the target component, it requires a liquid pump for each of the column for protein removal and the separation column for separation and analysis of the target component.
There was a problem in that the accuracy of analysis decreased due to the complicated device configuration.

In other words, in the various methods described above, the accuracy of separation 1 analysis of the target component bound to protein is low because there is no effective protein removal method for analyzing the target component by deproteinizing the protein bound to the target component. It wasn't enough.

[Purpose of the invention]

An object of the present invention is to provide a method and an apparatus for analyzing a target component, which can quickly, sufficiently, and easily remove proteins bound to the target component, and can therefore analyze the target component with high precision. be.

[Summary of the invention]

The present inventors have developed a giant reticular ion exchange resin (a copolymer of styrene divinylbenzene, mainly with a particle size of 3 to 100 μm).
We have conducted extensive studies on the interaction between microparticle gels with ion-exchange groups bound to albumin (a protein mainly found in biological fluids such as blood, which is bound to active substances in our daily lives). As a result, we obtained the following knowledge.

In other words, since proteins are ampholytes, their total charge changes depending on the pH; at a pH lower than the isoelectric point, it becomes positive and is adsorbed on a cation exchanger, and at a pH higher than the isoelectric point, it becomes positive.
Since it becomes negative with H, it is adsorbed on an anion exchanger. However, when a giant reticulated ion exchange resin is used as the ion exchanger, the adsorption of the protein increases by changing the pH. (2) Even if the ionic strength is set to 3 mot/L (Na CL ) or higher, (3) organic solvents such as acetone tril (
We have found that the above protein no longer elutes even when the concentration is increased to V/Vl. The same was true for cytochrome (molecular weight approximately 12,000 toltons) and myoglobin (molecular weight approximately 17,500 toltons), which have relatively small molecular weights as proteins.

The reason why proteins are not easily desorbed from the giant reticular ion exchange resin is thought to be because the proteins enter into the pores within the molecules of about 100 molecules within this resin.

Therefore, by using a giant reticulated ion exchange resin as a packing material, for example, in a protein adsorption column, the eluent's p.
By simply adjusting H, only the target component can be separated from the protein bound to the target component and analyzed.

The present invention has been made based on this knowledge. Specifically, the first invention of the present application includes a first step of bringing the protein into a charged state by adjusting the pH of a solution containing a test substance consisting of a protein bound to a target component, and a giant reticular ion exchange process. A second layer adsorbs the charged protein with a resin.
a third step of separating only the target component from the protein adsorbed by the giant reticular ion exchange resin; and a fourth step of analyzing the target component separated in the third step.
A second invention of the present application is a method for analyzing a target component, which is characterized by comprising steps of: a carrier liquid supply means to which a carrier liquid having a predetermined pH is supplied; and a protein bonded to the target component in the carrier liquid. a protein removal column charged with a giant reticular ion exchange resin that adsorbs proteins contained in the charged test substance according to a predetermined pH in the carrier liquid; An eluent supply means for supplying an eluent to the protein removal column to elute the target component from the protein adsorbed in the protein removal column, and an analysis means for analyzing the target component eluted by the eluent. This is a target component analysis device characterized by the following.

[Embodiments of the invention]

Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

FIG. 1 is an overall configuration diagram showing an embodiment of an apparatus for analyzing a target component, which is the second invention of the present application.

In the figure, storage tanks 8A, 8B, and 8C that store carrier liquid or eluent have piping 11, piping 12.
Piping 13 is connected. The pipes 12 and 13 are connected by a channel switching valve 15, and the pipe 12 to which the pipe 13 is connected is connected to the pipe 11 by a channel switching valve 5.

A pipe 14 is connected to this flow path switching valve 5,
This piping 14 is connected to a protein removal column 3 filled with a giant reticular ion exchange resin. A pump 1, a pressure gauge 9A, and an injection section 2 for injecting a sample are provided in the middle of the pipe 14. A piping 16 is connected to the protein removal column 3, and this piping 16 is connected to a separation column 4 for separating target components separated from proteins. 19. A pressure gauge 9B is connected to the pipe 16. A pipe 17 coming out of the separation column 4 is connected to a detector 6, and a recorder 7 for displaying analysis results is connected within the detector 6.

Next, the operation of this embodiment will be explained.

First, all flow path switching valves are closed and the pump 1 is driven to introduce carrier liquid from a carrier liquid storage tank (not shown) into the protein removal column 3. Next, from the sample injection section 2, the target component is bound and the test substance consisting of round white matter is supplied to the upstream side where the carrier liquid goes to the protein removal column 3.

The carrier liquid has a predetermined pH, and the proteins contained in the analyte are charged and adsorbed by the giant reticular ion exchange resin in the protein removal column 3 due to this pH. The giant reticular ion exchange resin is made by bonding ion exchange groups corresponding to the charged state of proteins to a microparticle gel made of a copolymer of styrene and divinylbenzene. For example, if the protein is positively charged, cation exchange groups need to be attached to styrene and divinylbenzene.

After the charged protein is adsorbed to the protein removal column 3, the flow path switching valve 15t is closed and the flow path switching valve 5 is opened, so that the eluent in the storage tank 8 is transferred to the protein removal column 3. so that it flows into

Next, after a predetermined period of time has elapsed, the flow path switching valve 15 is adjusted so that the eluent in the storage MsB flows in the same manner as the eluent in the storage tank 8A. Similarly, the eluent in the storage tank 8B is introduced into the protein removal column 3, and after a predetermined period of time has elapsed, the eluent in the storage tank 8B is
The eluent in C is introduced into the separation column instead of the eluent in reservoir 8B.

The target component bound to the protein adsorbed by the protein removal column 3 is separated from the protein by the eluent flowing into the column 3. After the target wood components separated from proteins are introduced into the separation column 4, the retention time corresponding to each target component is determined and analyzed. The retention time can be determined in advance using a standard sample.

Each target component is different depending on the liquid properties (pH) of the eluent.
Each target component is separated from the protein.

The separation column 4 is used for the purpose of increasing the separation ability of the target component, since the protein removal column 3 on which proteins are adsorbed is short. Therefore, this separation column 4 can be used for 6 types of chromatography.

Each target component separated by the separation column 4 is introduced into a detector 6, and a detection peak is detected for each target component. For example, an absorbance meter, a potentiometer, or the like can be used as the detector 6.

In this example, the eluent is introduced into the separation column 3 through three channel systems, but depending on the properties of the target components, it is possible to use a predetermined eluent and to adopt a predetermined flow channel system for the eluent. can.

Next, an experimental example of analysis using the analyzer according to the above embodiment will be explained.

(Experimental Example 1) In this experimental example, allopurinol present in serum bound to protein was analyzed.

The protein removal column 3 is a stainless steel chromatography tube with an inner diameter of 4 heads and a length of 1 cnt packed with a giant reticular anion exchange resin (Hitachi Gel◆3013-N), and the separation column 4 has an inner diameter of 4 heads and a length of 1 cnt.
6+aw, length 25 crn stainless steel chromato f
The column was packed with the same packing material used for the protein adsorption column. The packing material used in the separation column 4 is not limited to this example, and various packing materials can be used depending on the substance to be analyzed.

Approximately 1 μg of allopurinol and hyboxanthin in human serum 2
The test substance is dissolved in rr1t, and 20μt of it is
was injected through injection 2 and mixed into the carrier liquid, and then the proteins in the serum bound to allopurinol and hyboxanthin were adsorbed to the protein removal column 3, and allopurinol and the like were separated and analyzed.

Detection was performed by spectrophotometry at a wavelength of 250 nm. The eluent used was 0.001 mot/t for 8A.
Phosphate buffer solution (1) H6,Ol, 0.06 m for 8B
ol/ ammonium chloride - 0,01 mol/l - ammonium dihydrogen phosphate - 6% fV/V) acetonitrile mixture (pH 4,8), and for 8C, use one twice the concentration of the eluent of 8B. Using. The eluent flow rate is 0.8ml
/m, and after running 8 eluents for 8 minutes, then 10
Run the eluent at 8B for minutes, then run the eluent at 8C for 15 minutes. The analysis results are shown in FIG. 2(B). For comparison, the analysis results of hyboxanthin and allopurinol, which do not contain protein or protein, are shown in FIG. 2(A). Figure 2 (A
) and (B), there is no difference in the detected peaks, and it is clear that pretreatment for protein removal is unnecessary.

For reference, after repeatedly analyzing a serum sample several dozen times, we removed the packing materials from protein removal column 3 and separation column 4 and stained them with Fromcresol Green Reagent, a kit for quantitative analysis of serum proteins. Deproteinization column 3
Only the filling material was stained green. This fact revealed that the proteins in the serum were retained in the protein removal column 3 and did not reach the separation column 4.

(Experimental Example 2) The same protein removal column 3 used in Experimental Example 1 was filled with giant reticular cation exchange resin 1 millimeter gel ÷ 3013-8, and the separation column 4 was filled with a stainless steel column with an inner diameter of 4 W++ ms and a length of 15. A steel chromato tube filled with cation exchange resin Hitachi Custom Ion Exchange Resin 2619F was used. 10 μt of isolated plasma from a hepatitis patient was used as a sample and injected into the analyzer shown in the above example, and the amino acids contained therein were analyzed. The detection utilized the fluorescence reaction of orthophthalaldehyde (OPA), and the eluent used was an excitation wavelength of 360 nm and a fluorescence wavelength of 440 nm. The reagents shown in Table 1 were dissolved in 1 ton of distilled water.

Table 1 Elution composition for amino acid analysis The flow rates were O, tm//, and the mixing ratio of 8A and 8B was as shown in Table 2.

Table 2 Mixing ratio of eluent Note that in this experimental example, a cleaning solution for eluating the protein adsorbed by the protein removal column 3 is stored in the storage tank 8C. This cleaning solution is acetone with concentrated alkali added. The protein adsorbed by the protein removal column 3 cannot be eluted with low or medium concentration alkali.

The above OPA reagent has a flow rate of 0.6 m/-, 0.3
It was reacted with the eluate of separation column 4 at 35C in a coil of 3 ws IDX 3 m. The results are shown in FIG.

According to FIG. 3, various amino acids can be separated with high accuracy. In addition, in this experimental example, as in Experimental Example 1, a comparative test was conducted using a standard sample that does not contain Yu/Baku. The results of the comparative test are the same as those shown in FIG. 3, so illustration is omitted.

This experimental example also shows that proteins are completely removed by the protein removal column.

(Experimental Example 3) A column similar to the protein removal column used in Experimental Example 1 was used as a separation column, and a stainless steel chromatography tube with an inner diameter of 4.6 wx and a length of 25 cm was filled with silica gel Hitachi gel φ3 for reverse phase and distribution.
Using a sample filled with 053, gout patient plasma components were analyzed.

First, 20 μt of gout patient plasma was injected into the injection section 2 of the device shown in Fig. 1, and 50 m
ol/l phosphorus buffer (RIH6,0) at a flow rate of 1.0
m/- into column 3, and other storage tanks 8B and 8C.
A single elution method was used without using.

Detection was by spectrophotometry at 25Qnm. The results of the analysis are shown in Figure 4.

According to FIG. 4, it can be seen that each constituent contained in gout patient plasma has been analyzed with high accuracy. Therefore, like Experimental Example 2, this experiment is highly useful to clinical testing departments.

In addition, in this experimental example, a comparative test was also conducted using a standard sample that does not contain protein, but the results are the same as those shown in FIG. 4, so illustration is omitted.

Next, another embodiment of the analyzer will be described.

FIG. 5 is an overall configuration diagram showing another embodiment of the analyzer.

This embodiment is the same as the embodiment shown in FIG. 1, except that a differential pressure gauge 10 is installed to measure the pressure increase in the protein removal column 3. According to this embodiment, the state of deterioration of the protein adsorption column can be ascertained using a differential pressure gauge.

Similar to FIG. 5, FIG. 6 is an overall configuration diagram showing another embodiment of the analyzer.

This embodiment differs from the above embodiments in that the differential pressure #
Multi-way switching valves 21A and 21B are installed before and after the IO,
B is provided with a pump 22 for feeding the washing liquid (regenerating liquid) 24 described in Experimental Example 2 to the protein removal column 3.

When the differential pressure gauge 10 shows a predetermined value, the switching valve 21A
and 21B, and the regenerating liquid 24 is sent to the protein adsorption column 3 using the pump 22 for regeneration. According to this embodiment, there is an effect that the protein adsorption column 3 can be regenerated without being removed from the system.

FIG. 7 is an overall configuration diagram showing another embodiment of the analyzer.

This embodiment is different from the embodiment shown in FIG.
, and an automatic six-way switching valve 23 are provided, and the protein removal column 3, the regenerating liquid 24, and the regenerating liquid feeding pump 22 are connected through the automatic six-way switching valve 23. In a normal analysis, the eluent 24 passes through a flow path 71' shown by a broken line in the automatic six-way switching valve 23, passes through the protein removal column 3, and reaches the separation column 4. On the other hand, the control device (ii25 is a signal Sz 'i) which is used to repeat the analysis and outputs the signal to the automatic six-way switching valve 23 and the regenerating liquid feed pump 22 to drive them. At this time, the eluent is The flow path shown by the solid line in the valve 23 travels through the flow path and reaches the separation column 4 without passing through the protein removal column 3.Then, the regeneration liquid 24 is supplied by the regeneration liquid feed pump 22 which is reversely operated by the control device 25. is sent to the protein removal column 3, and this column 3 is regenerated.According to this embodiment, analysis and regeneration of the protein adsorption column can be performed automatically.

Note that the control of regeneration of the protein removal column shown in this example can also be applied to a conventional bulk column chromatography apparatus.

〔Effect of the invention〕

As explained above, according to the present invention, since a giant reticular ion exchange resin is used, proteins bound to a target component can be removed sufficiently and quickly from the target component. . Therefore, analysis of the target component can be performed quickly and sufficiently.

[Brief explanation of drawings]

FIG. 1 is an overall configuration diagram showing one embodiment of the analyzer according to the present invention, FIG. 2 is a graph showing the results of an analysis experiment using the analyzer according to the present invention, and FIG. 3 is an example of an analysis experiment for hepatitis patient plasma. Graphs showing the results, FIG. 4 is a graph showing the results of an analysis experiment on gout patient plasma, and FIGS. 5 to 7 are configuration diagrams showing an embodiment in which a differential pressure gauge 10 is provided in the protein removal column of the analyzer. be. 2... Injection part, 3... Protein removal column,
4... Separation column, 6... Analyzer, 8A, 8B,
8C... Eluent storage tank, 10... Differential pressure gauge.

Claims (1)

[Scope of Claims] 1. A first step in which the protein is brought into a charged state by adjusting the pH of a solution containing a test substance consisting of a protein bound to a target component, and a step in which the protein is charged with a giant reticular ion exchange resin a second step of adsorbing the protein in the state, a third step of separating only the target component from the protein adsorbed by the giant reticular ion exchange resin, and an analysis of the target component separated in the third step A method for analyzing a target component, comprising a fourth step. 2. A carrier liquid supply means for supplying a carrier liquid having a predetermined pH; a test substance supply means for supplying a test substance consisting of a protein bound to a target component to the carrier liquid; A protein removal column charged with a giant reticular ion exchange resin that adsorbs proteins contained in a test substance charged by the pH of An apparatus for analyzing a target component, comprising: an eluent supply means for supplying the eluent to the protein removal column; and an analysis means for analyzing the target component eluted by the eluent.